Albumin nanoparticles and uses thereof

Abstract

The present invention provides an albumin nanoparticle, comprising: a neoantigen peptide conjugated to an albumin hitchhiking compound to form a conjugated compound; and a plurality of albumins; wherein the conjugated compound is non-covalently bonded to the plurality of albumins via the albumin hitchhiking compound; wherein the plurality of albumins are non-covalently bonded and covalently crosslinked to each other; and wherein the conjugated compound is configured to be released from the albumin nanoparticle when subjected to pH 6 and below.

Description

[0001] Albumin Nanoparticles and Uses Thereof

[0002] Technical Field

[0003] The present invention relates, in general terms, to albumin-based nanoparticles and their uses thereof.

[0004] Background

[0005] Neoantigens are immunogenic peptides aberrantly expressed only in tumor cells, making them are unique targets for developing personalized cancer vaccines.

[0006] Unlike other immunotherapeutic approaches such as cell-based therapies (chimeric antigen receptor T-cells or dendritic cell (DC) vaccines), immunotherapy using neoantigen peptides are generally safe, easy to administer, highly immunogenic and elicit long-lasting effects in patients with solid tumors. However, despite its ability to generate robust antigen specific T- cells, clinical application of neoantigen vaccines is hampered by quick degradation of naked peptides in-vivo, resulting in ineffective antigen presentation and limited anti-tumor efficacy. Furthermore, the immunosuppressive microenvironment of the tumor, prevent tumor-homing and infiltration of T-cells induced by neoantigen vaccines.

[0007] These are the major limitations of neoantigen cancer vaccines in a therapeutic setting. Recent pre-clinical studies employed cationic liposomal (Amgen Inc.) and polymeric nano-systems (Avidea Technologies), to mitigate peptide stability issues. Although, these nanocarriers have several advantages (biocompatibility and immunostimulatory effects), they suffer in complex in-vivo conditions due to protein corona (PC) formation leading to aggregation, charge neutrality and size enlargement. Secondly, they are not adaptable to deliver peptides of diverse properties or involves multiple linker moieties and steps.

[0008] It would be desirable to overcome or ameliorate at least one of the above described problems.

[0009] Summary

[0010] The present disclosure provides an albumin nanoparticle, comprising : a) a neoantigen peptide conjugated to an albumin hitchhiking compound to form a conjugated compound; and b) a plurality of albumins; wherein the conjugated compound is non-covalently bonded to the plurality of albumins via the albumin hitchhiking compound; wherein the plurality of albumins are non-covalently bonded and covalently crosslinked to each other; and wherein the conjugated compound is configured to be released from the albumin nanoparticle when subjected to pH 6 and below.

[0011] In some embodiments, the neoantigen peptide is characterised by an amino acid chain length of 20 residues to 40 residues.

[0012] In some embodiments, the neoantigen peptide is characterised by an amino acid chain length of 25 residues to 30 residues.

[0013] In some embodiments, the neoantigen peptide comprises a shared hot spot mutation.

[0014] In some embodiments, the albumin hitchhiking compound is a fluorophore configured to be excitable under near-infrared (NIR) irradiation.

[0015] In some embodiments, the albumin hitchhiking compound is indocyanine green (ICG).

[0016] In some embodiments, the neoantigen peptide is conjugated to an albumin hitchhiking compound via an amide bond.

[0017] In some embodiments, the conjugated compound is characterised by a mole ratio of neoantigen peptide to albumin hitchhiking compound of 1 : 1 to 5: 1.

[0018] In some embodiments, the albumin nanoparticle is characterised by a percent loading of neoantigen peptide of about 2 %wt / wt to about 30 %wt / wt.

[0019] In some embodiments, the albumin nanoparticle is characterised by a percent loading of albumin hitchhiking compound of about 2 %wt / wt to about 30 %wt / wt.

[0020] In some embodiments, the conjugated compound is non-covalently bonded to the plurality of albumins via electrostatic interaction, van Der Waals forces, and / or hydrogen bonding.

[0021] In some embodiments, the neoantigen peptide is configured to be cleaved from the albumin hitchhiking compound when subjected to pH 6 and below.

[0022] In some embodiments, the plurality of albumins are non-covalently bonded to each other via hydrophobic interaction, hydrogen bonding, and / or a reduced solubility of albumin induced by a non-solvent.

[0023] In some embodiments, the crosslink is configured to cleave when subjected to pH 6 and below.

[0024] In some embodiments, the plurality of albumins are covalently crosslinked to each other via a glutaraldehyde linker.

[0025] In some embodiments, the plurality of albumins are covalently crosslinked to each other via their lysine residues thereof.

[0026] In some embodiments, the albumin nanoparticle is characterised by a percent crosslink of about 50% to about 80%.

[0027] In some embodiments, the albumin nanoparticle is characterised by a particle size of about 90 nm to about 400 nm. In some embodiments, the albumin nanoparticle is characterised by a particle size of about 150 nm.

[0028] In some embodiments, the albumin nanoparticle is characterised by a polydispersity index of less than about 0.4.

[0029] In some embodiments, the albumin nanoparticle is characterised by a zeta potential of about -10 mV to about -80 mV.

[0030] In some embodiments, the albumin nanoparticle is characterised by a zeta potential of about -45 mV.

[0031] The present disclosure also provides a pharmaceutical composition comprising the albumin nanoparticle as disclosed herein, and optionally an excipient.

[0032] The present disclosure also provides a method of synthesizing an albumin nanoparticle, comprising : a) conjugating a neoantigen peptide with an albumin hitchhiking compound to form a conjugated compound; b) mixing the conjugated compound with albumin in order for the conjugated compound to non-covalently bind to albumin; and c) nanoprecipitating and crosslinking the albumin of step b) in order to form the albumin nanoparticles.

[0033] The present disclosure also provides a method of delivering a neoantigen peptide to a subject in need thereof, comprising administering a therapeutic amount of the albumin nanoparticle as disclosed herein to the subject.

[0034] The present disclosure also provides a method of treating and / or preventing a cancer in a subject in need thereof, comprising administering a therapeutic amount of the albumin nanoparticle as disclosed herein to the subject.

[0035] In some embodiments, the cancer is selected from nasopharyngeal carcinoma, lung cancer, breast cancer, colorectal cancer, melanoma, and a combination thereof.

[0036] In some embodiments, the method further comprises a step of photothermally treating the cancer using near-infra red (NIR) irradiation.

[0037] Brief description of the drawings

[0038] Embodiments of the present invention will now be described, by way of nonlimiting example, with reference to the drawings in which:

[0039] Figure 1 : General scheme of synthesis of pH-sensitive albumin nanocarrier encapsulating neoantigen peptide coupled to NIR-dye ICG.

[0040] Figure 2: (A) Hydrodynamic size and zetapotential of peptide-ICG loaded albumin nanoparticles. (B) Effect of pH and temperature on the size of nanoparticles dispersed in PBS. (C) UV-Vis-NIR absorbance spectra of free ICG and peptide-ICG loaded albumin nanoparticles. (D) Effect of peptide charge on the size and loading efficiency of peptide-ICG in nanoparticles. (E) Effect of sonication on nanoparticle stability (F) Effect of long-term storage of nanoparticles at -20 degrees.

[0041] Figure 3: (A) Effect of Free-ICG at different concentration on MC38 and LLC1 cancer cells following 24 h incubation. (B) Effect of NIR light irradiation on the viability of MC38 and LLC1 cells following 6h incubation of free-ICG and peptide- ICG loaded albumin nanoparticles. (C) Confocal microscopy images showing localization of free-ICG and peptide-ICG loaded albumin nanoparticles in mouse BMDCs following 24 h incubation. (D) Singlet oxygen production following NIR irradiation. (E) Effect of NIR-PDT on the viability of MC38 cancer cells.

[0042] Figure 4: Representative live confocal laser scanning microscopic images of MC38 cells treated with f carboxy-H2DCFDA and different formulation with and without NIR light to determine the production of ROS.

[0043] Figure 5: (A) Uptake kinetics of free-ICG and Peptide-ICG loaded albumin nanoparticles in MC38 cancer cells following different incubation times. (B) Localization of Free-ICG and Peptide-ICG loaded albumin nanoparticles following 24 h incubation. Yellow arrows pointing at colocalization of ICG fluorescence in the cell surface.

[0044] Figure 6: Confocal laser scanning microscopy images showing (A) Localization of internalized ICG, Peptide-ICG and Peptide-ICG albumin nanoparticles in JAWSII mouse DCs following 3 h incubation. (B) Immunofluorescent staining of anti-H-2 Kb loaded with SINFEKL.

[0045] Figure 7: (A) In-vivo determination of antigen specific T-cells using H-2 Kb SIINFEKL dextramer by flowcytometry. (B) Flowcytometry analysis of BMDCs maturation following treatment with various peptide formulations. (C) Representative images of hemolysis assay following treatment of nanoparticles with mouse RBCs and percent hemolysis. (D) Flowcytometric analysis of H-2 Kb SIINFEKL dextramer positive CD8+ antigen specific T-cells in mice immunized with free-peptide or peptide-ICG loaded albumin nanoparticles.

[0046] Figure 8: Detection of antigen-specific T-cells against MS-identified TS / TAA candidates using intracellular cell staining (ICCS) in peptide-stimulated T-cell cultures. Peptide 2, presented on HLA-A2:01, is among the top TS / TAA candidates identified from the NPC cell line C17, but not on immortalized healthy nasopharyngeal epithelial cells NP69. PBMCs expressing HLA-A*02:01 were stimulated with vehicle alone (Grey; DMSO only) in all dot plots, LMP2A peptides only (Black; left column), and Peptide 2 peptides only (Black; right column) along with cytokines for 2 weeks. Cells were then starved overnight without cytokines and then rechallenged with no peptide, PMA only, LMP2A only, or peptide 2 only for 6 h before ICCS staining. Representative dot plots show fluorescence intensities representing intracellular staining of Interferon-gamma (IFNy) on the y-axis. Each dot represents one cell, and the red values on top indicate percentages of IFNy+-CD8+T-cells (top row; CTL) and CD4+ T cells.

[0047] Detailed description

[0048] Without wanting to be bound by theory, in order to overcome at least one of the limitations, the inventors developed self-assembled pH-sensitive multifunctional nanoparticle comprising neoantigen. The neoantigen peptide is chemically conjugated to "albumin hitchhiker" such as indocyanine green (ICG). The conjugation of neoantigen peptide to a near-infrared (NIR) light excitable dye indocyanine green (ICG) imparts multiple functionalities such as peptide tracking and NIR light activated photodynamic induction of reactive oxygen species, that could reverse the immunosuppressive tumor microenvironment. The conjugated peptide may then be formed into an albumin-based nanocarrier, which may dissociate at certain pH to release the ICG-peptide (Figure 1). The albumin-based nanocarrier may be used as a nanovaccine, which protects neoantigen peptides thus improve stability, prolong its circulation half-life, enable pH-dependent escape of the peptide from acidic endosome to cytosol and target the cargo directly to lymph nodes. When utilized with near-infrared photodynamic therapy (NIR-PDT), the releases of neoantigen peptide at the tumor microenvironment is accelerated. Further, with NIR-PDT, ICG is induced to activate thus triggering immunological tumor cell death, epitope spreading and reverse the immunosuppressive tumor microenvironment. This combination of neoantigen vaccine and photodynamic therapy (photoimmunotherapy) may enhance the anti-tumor effects synergistically on both primary tumors and distant metastatic lesions, triggering a robust, long-lasting and tumor-specific immune response in cancer patients.

[0049] The albumin nanoparticle is multifunctional. For example, the albumin nanoparticle may comprise an FDA-approved NIR-dye ICG, which has a natural affinity for albumin, and reversibly binds to albumin through non-covalent linkages. ICG not only acts as an albumin binder, but it may also be a tracker to study the transport and localization of the peptide, and is NIR-excitable to photodynamic / photothermal therapy (PDT / PTT) agent that can be excited to produce cytotoxic reactive oxygen species (ROS), even in the absence of oxygen. The albumin nanoparticle provides targeted delivery to lymph nodes (LN). For example, the presence of albumin hitch-hiker ICG, increases the size of the peptides by many folds. Large size of albumin (66 kDa) forces the peptides to be disseminated through lymphatics to draining LNs. This offers a longer window for albumin nanoparticles to modulate lymphocytes in LNs, be internalized by DCs, facilitating effective antigen presentation.

[0050] Further, the albumin nanoparticle is pH sensitive. For example, glutaraldehyde used to crosslink the albumin through the interaction between aldehyde and amine residues, results in pH-sensitive Schiff base bond. Though the nanoparticles are extremely stable at pH>6 even with sonication and at 37 degree Celsius, they are unstable at acidic pH leading to swelling of the nanoparticles at acidic pH. This enables pH controlled release of the peptide from the endo-lysosomal pathway, for efficient antigen presentation.

[0051] In contrast, previously reported albumin-peptide fusion and in-vivo albumin binding Evan's blue-peptide fusions, are not preformed albumin nanocarriers. These form nanoaggregates following administration in to the in-vivo environment. Preformed nanocarriers will have minimal characteristic changes in-vivo.

[0052] The albumin nanoparticles are size-tunable. Loading and particle size may be controlled by tuning the molar ratios of the albumin to conjugated compound in the reaction mixture.

[0053] The albumin nanoparticles are biocompatible and biodegradable. The nanoparticle is predominantly composed of albumin which is the most abundant protein in human body and hence is extremely biocompatible and biodegradable, and least toxic compared to other polymeric and inorganic nanoparticles. If ICG is used, it is FDA approved and already in clinical use.

[0054] The albumin nanoparticle is synthesised via a versatile approach as the method is based on conjugation chemistry. Any neoantigen peptide may be coupled with albumin hitchhiking compound irrespective of its charge or hydrophobicity, unlike liposomal or polymeric carriers. The flanking sequences of the peptides can be easily modified to carry 5-6 lysine residues amenable for effective albumin hitchhiking compound conjugation. The process is facile and scalable, in which a 2-step nanoparticle synthesis protocol enables highly reproducible and scalable production of the nanoparticles. The entire fabrication process may be automated to achieve high quality, precisely controlled, reproducible nanoparticles.

[0055] Accordingly, the present disclosure provides an albumin nanoparticle, comprising: a) a neoantigen peptide conjugated to an albumin hitchhiking compound to form a conjugated compound; and b) a plurality of albumins; wherein the conjugated compound is non-covalently bonded to the plurality of albumins via the albumin hitchhiking compound; wherein the plurality of albumins are non-covalently bonded and covalently crosslinked to each other; and wherein the conjugated compound is configured to be released from the albumin nanoparticle when subjected to pH 6 and below.

[0056] Without wanting to be bound by theory, forming albumin nanoparticles via both nanoprecipitation and cross-linking is advantageous as the neoantigen peptides may be configured to be only released at a tumor site, which are often characterised by an acidic microenvironment at pH 6 or below. The albumin nanoparticles maintains its size, facilitating its uptake into tumor cells via enhanced permeability and retention effect (EPR).

[0057] The term "antigen" as used herein is a substance that induces an immune response.

[0058] As used herein the term "neoantigen" is an antigen that has at least one alteration that makes it distinct from the corresponding wild-type, parental antigen, e.g., via mutation in a tumor cell or post-translational modification specific to a tumor cell. A neoantigen can include a polypeptide sequence or a nucleotide sequence that is produced by cancer cells and is not present in normal, healthy cells. A mutation can include a frameshift or non-frame shift indel, missense or nonsense substitution, splice site alteration, genomic rearrangement or gene fusion, or any genomic or expression alteration giving rise to a neo-ORF. A mutations can also include a splice variant. Post- translational modifications specific to a tumor cell can include aberrant phosphorylation. Post-translational modifications specific to a tumor cell can also include a proteasome-generated spliced antigen. In this regard, neoantigens are foreign peptides or proteins that are absent in normal tissues, but can arise from tumors through various mechanisms, such as genomic mutation, aberrant transcriptomic variants, post-translational modifications (PTMs), and viral ORFs. Neoantigen may thus be recognised by the body's immune system as foreign or abnormal, which may trigger an immune response against the cancer cells.

[0059] Examples of neoantigens are disclosed in Liepe et al., A large fraction of HLA class I ligands are proteasome-generated spliced peptides; Science. 2016 Oct 21; 354(6310); 354-358; Xie, N. et al., Neoantigens: promising targets for cancer therapy. Sig Transduct Target Ther 2023; 8, 9; Tao Wu et al., Neodb: a comprehensive neoantigen database and discovery platform for cancer immunotherapy, Database, Volume 2023, 2023, baad041; and Pearlman AH et al., Targeting public neoantigens for cancer immunotherapy Nat Cancer. 2021; 2(8):865-867; Nat Cancer. 2021; 2(5):487-497, the references of which are incorporated herein.

[0060] As used herein the term "NEO-ORF" is a tumor-specific ORF arising from a mutation or other aberration such as splicing.

[0061] By a "polypeptide" or "peptide" is meant a polypeptide that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally- occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide. An isolated polypeptide may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0062] In some embodiments, the neoantigen peptide is characterised by an amino acid chain length of 5 residues to 50 residues. In some embodiments, the neoantigen peptide is characterised by an amino acid chain length of 10 residues to 50 residues, 15 residues to 50 residues, 20 residues to 50 residues, 20 residues to 45 residues, 20 residues to 40 residues. In some embodiments, the neoantigen peptide is characterised by an amino acid chain length of 20 residues to 40 residues. In some embodiments, the neoantigen peptide is characterised by an amino acid chain length of 25 residues to 30 residues.

[0063] In some embodiments, the neoantigen peptide binds to major histocompatibility complex (MHC) class I and / or II. In some embodiments, neoantigenic peptide binds to human leukocyte antigen (HLA) -A, -B, -C, -DP, -DQ, and / or -DR.

[0064] In some embodiments, the neoantigen peptide is characterised by a binding affinity of less than about 1000 nM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, or about 300 nM.

[0065] In some embodiments, the neoantigen peptide comprises a shared hot spot mutation. Shared hot spot mutation refer to specific genetic mutation that is found across different types of cancer. These mutations occur at specific locations or "hot spots" in the DNA sequence and are often associated with the development and progression of cancer.

[0066] These type of mutations have high recurrence, and are frequently observed in multiple cancer types. Shared hot spot mutations often occur in genes that are known to be important in regulating cell growth, survival, and differentiation, such as KRAS, BRAF, and TP53. Examples of shared hot spot mutations include KRAS codon 12 and 13 mutation, BRAF 600E mutation, TP53 R175H mutation, PIK3CA mutation, and RAS mutation.

[0067] In some embodiments, the neoantigen peptide is selected from Table 1.

[0068] Table 1. Examples of neoantigen peptides

[0069]

[0070] Human Leukocyte Antigen (HLA) molecules may exhibit considerable binding promiscuity, enabling them to present a diverse array of peptides to T cells. As a result, a single HLA allele may bind multiple distinct 9mer peptides, thereby enhancing the breadth of immune surveillance at the individual level.

[0071] Conversely, the same 9mer peptide may be presented by multiple HLA alleles, particularly among closely related variants that share similar peptide-binding grooves. This may facilitate cross-presentation across genetically diverse individuals. This may lead to more effective immune responses.

[0072] In some embodiments, the neoantigen peptide is selected from KRAS-G12D peptide (derived from a common mutation in the KRAS gene, which is found in many types of cancer, including colorectal, lung, and pancreatic cancers), TP53- R175H peptide (associated with a mutation in the TP53 gene, which is a commonly mutated gene in various cancers), BCR-ABL peptide (derived from the fusion of the BCR and ABL genes, which is a hallmark of chronic myeloid leukaemia), EGFR-L858R peptide (associated with a mutation in the EGFR gene, which is commonly found in non-small cell lung cancer), NY-ESO-1 peptide (derived from the NY-ESO-1 cancer-testis antigen, which is expressed in a variety of cancers, including melanoma, ovarian, and lung cancer), and MUC16 peptide (associated with mutations in the MUC16 gene, which is commonly found in ovarian and pancreatic cancers).

[0073] In some embodiments, the neoantigen peptide comprises a linker at its C- and / or N-terminus. In some embodiments, the neoantigen peptide comprises one C- and / or N-terminal flanking amino acid chain. The flanking amino acid chain may comprise 2 to 10 lysine residues. The linker or flanking amino acid chain may comprise a bond which is cleavable at pH 6 and below. The linker may link the neoantigen peptide to the albumin hitchhiking compound.

[0074] In some embodiments, the linker may comprise hydrazone linkage, acetal / ketal linkage, imine (Schiff base) linkage, and / or ortho ester linkage.

[0075] "Hitchhiking" in the context of cargo delivery, refers to using an external carrier to transport cargo to its desired destination. Hitchhiking leverages the natural ability of proteins to navigate the vasculature while evading immune system clearance and perform specific tasks such as delivering nutrients to tissues, eliminating pathogens, and surveilling the immune system. The hitchhiker may interact with the carrier via non-specific adsorption driven by electrostatic interaction, van Der Waals forces, and hydrogen bonding; and / or site-specific adsorption driven by ligand-receptor interactions. The albumin hitchhiking compound is a compound which may hitchhike on albumin via at least one of the above mentioned interactions.

[0076] In some embodiments, the albumin hitchhiking compound is a fluorophore configured to be excitable under near-infrared (NIR) irradiation.

[0077] A fluorophore is a compound that may absorb light energy in the electromagnetic spectrum and then re-emit the energy as light of a different wavelength, in a process known as fluorescence. A fluorophore may be configured to be excitable under NIR irradiation is a fluorophore that may absorb light in the NIR region of the electromagnetic spectrum, typically between about 700 nm to about 1100 nm, and then emit light at a longer wavelength. The fluorophore may have an absorption spectrum that overlaps with the NIR region and an emission spectrum at a longer wavelength, which may be in the NIR region or the infrared region. The fluorophore may be a cyanine dye or a phthalocyanine. The fluorophore may be indocyanine green (ICG), Cyanine-7 (Cy7), heptamethine cyanine dyes (IR-783, IR-780, IR-775), or zwitterionic NIR fluorophore (ZW800-1).

[0078] In some embodiments, the albumin hitchhiking compound is a cyanine dye. In some embodiments, the albumin hitchhiking compound is indocyanine green (ICG).

[0079] The use of a fluorophore allows the utilization of the optical absorption characteristics in the near-infrared region to assist in peptide tracking. Further, NIR light-activated photodynamic induction of reactive oxygen species by the fluorophore aids in improving the tumor microenvironment; thereby improving the actual therapeutic effect achieved by the tumor vaccine peptides.

[0080] In some embodiments, the neoantigen peptide is conjugated to an albumin hitchhiking compound via an amide bond. For example, the neoantigen peptide may be conjugated to the albumin hitchhiking compound via EDC-NHS chemistry. The neoantigen peptide may be linked via a linker. The linker may comprise hydrazone linkage, acetal / ketal linkage, imine (Schiff base) linkage, and / or ortho ester linkage.

[0081] In some embodiments, the conjugated compound is characterised by a mole ratio of neoantigen peptide to albumin hitchhiking compound of about 1 : 1 to about 5: 1. In other embodiments, the mole ratio is about 1 : 1 to about 4: 1, about 1 : 1 to about 3: 1, about 1 : 1 to about 2: 1, about 2: 1 to about 5: 1, about 2: 1 to about 4: 1, about 2: 1 to about 3: 1, about 3: 1 to about 5: 1, about 3: 1 to about 4: 1, or about 4: 1 to about 5: 1. In some embodiments, the conjugated compound is characterised by a mole ratio of neoantigen peptide to albumin hitchhiking compound of about 1 : 1.

[0082] In some embodiments, the conjugated compound binds to albumin via non- covalent bonding. In some embodiments, the albumin hitchhiking compound binds to albumin via non-covalent bonding. In some embodiments, the albumin hitchhiking compound binds to albumin via electrostatic interaction, van Der Waals forces, and / or hydrogen bonding.

[0083] The interaction of the albumin hitchhiking compound with albumin (or human serum albumin (HSA)) depends on the values of R (R is defined as the molar ratio of albumin to albumin hitchhiking compound). The interaction of albumin hitchhiking compound with albumin may form two complexes with intrinsic binding constants (Ka) of 2.97X105(R<2) and 2.63xl04(R>2), respectively. The fluorescence and induced CD (ICD) spectra of albumin hitchhiking compound demonstrate that binding the first mole of albumin to albumin hitchhiking compound can form a chiral albumin hitchhiking compound-albumin complex with strong fluorescence emission, and the chirality and fluorescence of albumin hitchhiking compound-albumin complex can be significantly reduced by adding another mole of albumin to albumin hitchhiking compound. Furthermore, although both albumin hitchhiking compound and albumin hitchhiking compound-albumin complexes followed an energy-dependent endocytosis process to enter living cells, the cellular uptaken dynamic mechanism of albumin hitchhiking compound was significantly affected by the albumin conjugation.

[0084] The complexes may form through non-covalent binding of the albumin hitchhiking compound (ICG) to albumin, involving hydrophobic interactions, hydrogen bonding, and electrostatic forces.

[0085] Due to the large molecular size of albumin (66 kDa), complexation with the neoantigen peptide-conjugated albumin hitchhiking compound may direct the nanoparticle preferentially into the lymphatic system rather than through rapid vascular clearance. This mechanism may prolong the peptide's half-life in circulation and may enhance the likelihood of interaction with dendritic cells via albumin-binding receptors and caveolae-mediated endocytosis, thereby promoting antigen presentation, improved biodistribution, and enhanced immune activation compared to free peptides.

[0086] For vaccine delivery, a combination of strong (high Ka) and weak (low Ka) binding interactions is typically preferred. Strong binding may ensure complex stability and lymph node targeting, whereas weaker binding may allow for controlled release of the peptide payload, facilitating effective immune activation. Although there is no universally fixed optimal ratio, moderate-to- strong binding affinities are generally preferred for balancing delivery and release.

[0087] The release of the neoantigen peptide may be facilitated by acidic pH at the target site. Hence an albumin hitchhiking compound-albumin complex with a stronger binding interaction (higher Ka) may be preferred for better stability and lymphatic transport. For example, an albumin hitchhiking compound-albumin complex with a Kaof about 2.97X105may be preferred.

[0088] A lower molar ratio of albumin to albumin hitchhiking compound may result in stronger binding between the albumin hitchhiking compound and the albumin. This may cause an increase in fluorescence and chirality of the albumin hitchhiking compound-albumin complex due to tighter interaction. The stronger binding between the albumin hitchhiking compound and the albumin may be preferred initially for stability and lymphatic transport, while subsequent release of the neoantigen peptide may occur at the target site, such as the acidic tumor environment, facilitated by acidic pH or photoactivation, even if the initial complex is strongly bound. For example, an albumin hitchhiking compoundalbumin complex with a Kaof about 2.97X105may be preferred.

[0089] In some embodiments, the albumin nanoparticle is characterised by a percent loading of neoantigen peptide of about 2 %wt / wt to about 30 %wt / wt. In other embodiments, the percent loading is about 2 %wt / wt to about 25 %wt / wt, about 2 %wt / wt to about 20 %wt / wt, about 2 %wt / wt to about 15 %wt / wt, about 2 %wt / wt to about 10 %wt / wt, about 2 %wt / wt to about 5 %wt / wt, about 5 %wt / wt to about 30 %wt / wt, about 5 %wt / wt to about 25 %wt / wt, about 5 %wt / wt to about 20 %wt / wt, about 5 %wt / wt to about 15 %wt / wt, about 5 %wt / wt to about 10 %wt / wt, about 10 %wt / wt to about 30 %wt / wt, about 10 %wt / wt to about 25 %wt / wt, about 10 %wt / wt to about 20 %wt / wt, about 10 %wt / wt to about 15 %wt / wt, about 15 %wt / wt to about 30 %wt / wt, about 15 %wt / wt to about 25 %wt / wt, about 15 %wt / wt to about 20 %wt / wt, about 20 %wt / wt to about 30 %wt / wt, about 20 %wt / wt to about 25 %wt / wt, or about 25 %wt / wt to about 30 %wt / wt.

[0090] In some embodiments, the albumin nanoparticle is characterised by a percent loading of albumin hitchhiking compound of about 2 %wt / wt to about 30 %wt / wt. In other embodiments, the percent loading is about 2 %wt / wt to about 25 %wt / wt, about 2 %wt / wt to about 20 %wt / wt, about 2 %wt / wt to about 15 %wt / wt, about 2 %wt / wt to about 10 %wt / wt, about 2 %wt / wt to about 5 %wt / wt, about 5 %wt / wt to about 30 %wt / wt, about 5 %wt / wt to about 25 %wt / wt, about 5 %wt / wt to about 20 %wt / wt, about 5 %wt / wt to about 15 %wt / wt, about 5 %wt / wt to about 10 %wt / wt, about 10 %wt / wt to about 30 %wt / wt, about 10 %wt / wt to about 25 %wt / wt, about 10 %wt / wt to about

[0091] 20 %wt / wt, about 10 %wt / wt to about 15 %wt / wt, about 15 %wt / wt to about

[0092] 30 %wt / wt, about 15 %wt / wt to about 25 %wt / wt, about 15 %wt / wt to about

[0093] 20 %wt / wt, about 20 %wt / wt to about 30 %wt / wt, about 20 %wt / wt to about

[0094] 25 %wt / wt, or about 25 %wt / wt to about 30 %wt / wt.

[0095] In some embodiments, the albumin nanoparticle is characterised by a mole ratio of neoantigen peptide to albumin of about 5:1 to about 1:20. In other embodiments, the mole ratio is about 5:1 to about 1:15, about 5:1 to about 1:10, about 5:1 to about 1:5, about 5:1 to about 1:1, about 1:1 to about 1:20, about 1 : 1 to about 1:15, about 1 : 1 to about 1 : 10, about 1 : 1 to about 1 : 5, about 1:5 to about 1:20, about 1:5 to about 1:15, about 1:5 to about 1:10, about 1:10 to about 1:20, about 1:10 to about 1:15, or about 1:15 to about 1:20.

[0096] In some embodiments, the albumin nanoparticle is characterised by a mole ratio of albumin hitchhiking compound to albumin of about 1:1 to about 1:20. In other embodiments, the mole ratio is about 1:1 to about 1:15, about 1:1 to about 1 : 10, about 1 : 1 to about 1 : 5, about 1 : 5 to about 1 : 20, about 1 : 5 to about 1 : 15, about 1 :5 to about 1 : 10, about 1 : 10 to about 1 :20, about 1 : 10 to about 1 : 15, or about 1 : 15 to about 1 :20. In some embodiments, the mole ratio is about 1 : 1 to about 1 :5.

[0097] In some embodiments, the neoantigen peptide is encapsulated by the plurality of albumins. In some embodiments, the albumin hitchhiking compound is encapsulated by the plurality of albumins. The plurality of albumins thus forms a shell, or a protective layer, of the albumin nanoparticle.

[0098] In some embodiments, the neoantigen peptide is configured to be cleaved from the albumin hitchhiking compound when subjected to pH 6 and below. For example, the neoantigen peptide may be conjugated to the albumin hitchhiking compound via hydrazone linkage, acetal / ketal linkage, imine (Schiff base) linkage, and / or ortho ester linkage.

[0099] In some embodiments, the plurality of albumins are non-covalently bonded to each other via nanoprecipitation. In some embodiments, the plurality of albumins are non-covalently bonded to each other via hydrophobic interaction, hydrogen bonding, and / or a reduced solubility of albumin induced by a nonsolvent such as ethanol.

[0100] In some embodiments, the plurality of albumins are covalently bonded to each other via a crosslinker. The crosslinker may be glutaraldehyde. In some embodiments, the plurality of albumins are covalently crosslinked to each other via their lysine residues thereof.

[0101] In some embodiments, the crosslinker is cleavable when subjected to pH 6 or below. For example, the crosslinker may comprise hydrazone linkage, acetal / ketal linkage, imine (Schiff base) linkage, and / or ortho ester linkage.

[0102] Without wanting to be bound by theory, this causes the albumin nanoparticles to only be cleaved and dissociated near or at tumor cells, which are often characterised by an acidic microenvironment. Accordingly, the release of the neoantigen peptide may be improved.

[0103] In some embodiments, the albumin nanoparticle is characterised by a percent crosslink of about 50% to about 80%. In other embodiments, the percent crosslink is about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 60% toa bout 80%, about 60% to about 75%, about 60% to about 70%, about 60% to about 65%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%, about 70% to about 80%, about 70% to about 75%, or about 75% to about 80%. In other embodiments, the percent crosslink is about 60%.

[0104] In some embodiments, the albumin nanoparticle is characterised by a particle size of about 90 nm to about 400 nm. In other embodiments, the particle size is about 90 nm to about 350 nm, about 90 nm to about 300 nm, about 90 nm to about 250 nm, about 90 nm to about 200 nm, about 90 nm to about 150 nm, about 150 nm to about 400 nm, about 150 nm to about 350 nm, about 150 nm to about 300 nm, about 150 nm to about 250 nm, about 150 nm to about 200 nm, about 200 nm to about 400 nm, about 200 nm to about 350 nm, about 200 nm to about 300 nm, about 200 nm to about 250 nm, about 250 nm to about 400 nm, about 250 nm to about 350 nm, about 250 nm to about 300 nm, about 300 nm to about 400 nm, about 300 nm to about 350 nm, or about 350 nm to about 400 nm. In some embodiments, the albumin nanoparticle is characterised by a particle size of about 150 nm.

[0105] In some embodiments, the albumin nanoparticle is characterised by a polydispersity index of less than about 0.4. In other embodiments, the polydispersity index is less than about 0.3, less than about 0.2, or less than about 0.1.

[0106] In some embodiments, the albumin nanoparticle is characterised by a zeta potential of about -10 mV to about -80 mV. In other embodiments, the zeta potential is about -10 mV to about -60 mV, about -10 mV to about -40 mV, about -10 mV to about -20 mV, about -20 mV to about -80 mV, about -20 mV to about -60 mV, about -20 mV to about -40 mV, about -40 mV to about -80 mV, about -40 mV to about -60 mV, or about -60 mV to about -80 mV. In some embodiments, the albumin nanoparticle is characterised by a zeta potential of about -45 mV.

[0107] The present disclosure also provides a pharmaceutical composition comprising the albumin nanoparticle as disclosed herein, and optionally an excipient.

[0108] The present disclosure also provides a method of synthesizing an albumin nanoparticle, comprising : a) conjugating a neoantigen peptide with an albumin hitchhiking compound to form a conjugated compound; b) mixing the conjugated compound with albumin in order for the conjugated compound to non-covalently bind to albumin; and c) nanoprecipitating and crosslinking the albumin of step b) in order to form the albumin nanoparticles.

[0109] The present disclosure also provides a method of delivering a neoantigen peptide to a subject in need thereof, comprising administering a therapeutic amount of the albumin nanoparticle as disclosed herein to the subject.

[0110] The present disclosure also provides an albumin nanoparticle as disclosed herein for use in delivering a neoantigen peptide to a subject in need thereof.

[0111] The present disclosure also provides a use of an albumin nanoparticle as disclosed herein in the manufacture of a medicament for delivering a neoantigen peptide.

[0112] The present disclosure also provides a method of treating and / or preventing a cancer in a subject in need thereof, comprising administering a therapeutic amount of the albumin nanoparticle as disclosed herein to the subject. The present disclosure also provides an albumin nanoparticle as disclosed herein for use in treating and / or preventing a cancer in a subject in need thereof.

[0113] The present disclosure also provides a use of an albumin nanoparticle as disclosed herein in the manufacture of a medicament for treating and / or preventing a cancer.

[0114] In some embodiments, the cancer is characterised by a hotspot mutation epitope. In some embodiments, the cancer is a cancer tumor. In some embodiments, the cancer is selected from nasopharyngeal carcinoma, lung cancer, breast cancer, colorectal cancer, melanoma, and a combination thereof.

[0115] In some embodiments, the method further comprises a step of photothermally treating the cancer using near-infra red (NIR) irradiation. The NIR irradiation may have a wavelength of about 750 nm to about 900 nm, or preferably about 800 nm.

[0116] The present disclosure also provides a method of inducing an immune response in a subject in need thereof, comprising administering a therapeutic amount of the albumin nanoparticle as disclosed herein to the subject.

[0117] The present disclosure also provides an albumin nanoparticle as disclosed herein for use in inducing an immune response in a subject.

[0118] The present disclosure also provides a use of an albumin nanoparticle as disclosed herein in the manufacture of a medicament for inducing an immune response in a subject.

[0119] The immune response may be against cancer.

[0120] As used herein, the terms "prevent", "preventing" or "prophylactic" and the like, refer to reducing the probability of developing a disease or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease or condition. The terms "treat", "treating" or "treatment" and the like are meant to refer to reducing or ameliorating a disorder and / or symptoms associated therewith (e.g., a neoplasia or tumor). "Treating" may refer to administration of the therapy to a subject after the onset, or suspected onset, of a cancer. "Treating" includes the concepts of "alleviating", which refers to lessening the frequency of occurrence or recurrence, or the severity, of any symptoms or other ill effects related to a cancer and / or the side effects associated with cancer therapy. The term "treating" also encompasses the concept of "managing" which refers to reducing the severity of a particular disease or disorder in a patient or delaying its recurrence, e.g., lengthening the period of remission in a patient who had suffered from the disease. It is appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

[0121] The albumin nanoparticle may be administered to a subject as a pharmaceutically acceptable salt thereof. Suitable pharmaceutically acceptable salts include, but are not limited to salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

[0122] Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium. In particular, the present invention includes within its scope cationic salts eg sodium or potassium salts, or alkyl esters (eg methyl, ethyl) of the phosphate group. Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.

[0123] The albumin nanoparticle, or a pharmaceutically acceptable salt, or solvate thereof is administered to the patient in a therapeutically effective amount. As used herein, a therapeutically effective amount is intended to include at least partially attaining the desired effect, or delaying the onset of, or inhibiting the progression of, or halting or reversing altogether the onset or progression of macular degeneration.

[0124] The term "therapeutic effect" refers to some extent of relief of one or more of the symptoms of a disorder (e.g., a neoplasia or tumor) or its associated pathology. "Therapeutically effective amount" as used herein refers to an amount of an agent which is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survivability of the patient with such a disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying, and the like beyond that expected in the absence of such treatment. "Therapeutically effective amount" is intended to qualify the amount required to achieve a therapeutic effect. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the "therapeutically effective amount" (e.g., ED50) of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in a pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0125] As used herein, the term "effective amount" relates to an amount of compound which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.

[0126] Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the severity of the condition as well as the general age, health and weight of the patient to be treated.

[0127] The albumin nanoparticle may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition. The formulation of such compositions is well known to those skilled in the art. The composition may contain any suitable carriers, diluents or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.

[0128] The carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

[0129] The compound may be injected directly to the eye, and in particular the vitreous of the eye. The compound, composition or combination of the invention can be administered to the vitreous of the eye using any intravitreal or transscleral administration technique. For example, the compound, composition or combination can be administered to the vitreous of the eye by intravitreal injection. Intravitreal injection typically involves administering an albumin nanoparticle or a pharmaceutically acceptable salt, or solvate in a total amount between 0.1 ng to 10 mg per dose.

[0130] The albumin nanoparticle may also be suitable for intravenous administration.

[0131] Injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion. Carriers can include, for example, water, saline (e.g., normal saline (NS), phosphate- buffered saline (PBS), balanced saline solution (BSS)), sodium lactate Ringer's solution, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances, such as wetting or emulsifying agents, buffers, and the like can be added. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. By way of example, the compound, composition or combination can be dissolved in a pharmaceutically effective carrier and be injected into the vitreous of the eye with a fine gauge hollow bore needle (e.g., 30 gauge, 1 / 2 or 3 / 8 inch needle) using a temporal approach (e.g., about 3 to about 4 mm posterior to the limbus for human eye to avoid damaging the lens).

[0132] A person skilled in the art will appreciate that other means for injecting and / or administering the albumin nanoparticle can also be used. These devices and methods can include, for example, intravitreal or intravenous medicine delivery devices, and biodegradable polymer delivery members that are inserted in a human body or eye for long term delivery of medicaments.

[0133] It should be understood that in addition to the active ingredients particularly mentioned above, the composition this invention may include other agents conventional in the art having regard to the type of composition or combination in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and / or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of Wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

[0134] Examples

[0135] General

[0136] A method for a facile synthesis of a nanocarrier is described. The nanocarrier may protect and efficiently deliver neoantigen peptide-based cancer vaccines to lymph nodes. The nanocarrier is mainly composed of biocompatible and biodegradable albumin shell. The core of the nanocarrier consist of the payload which are the neoantigen peptides (25-30 amino acids long), conjugated with FDA approved NIR-light excitable dye ICG. Here, ICG binds albumin molecules together. Additionally, ICG gives the nanocarrier multiple functionalities such as it can be used to track the transport and localization of the peptide vaccines. Additionally, it can also be used to trigger NIR-light induced photodynamic killing of the cancer cells in a very tumor-targeted fashion. This albumin based multifunctional nanocarrier, due to its size, will be targeted to the lymph nodes, where it will be effectively internalized by the dendritic cells (DCs). Inside the acidic endosomes of the DCs, the pH-sensitive nanocarrier will swell up, releasing its contents where the peptide is further trimmed and loaded on the major histocompatibility complexes (MHCs). The MHCs with the loaded peptides will then be presented on the cell surface, allowing efficient activation antigen specific CD8+ T-cells. As the ICG released from the trimmed peptides accumulate in the tumor, external NIR light irradiation of the tumors could activate ICG to produce cytotoxic singlet oxygen. This would trigger immunologic cell death and epitope spreading to initiate a stronger recruitment of activated T-cells to the tumor, thus helping to overcome the immunosuppressive tumor micro- environment.

[0137] Nanoparticle synthesis and characterization

[0138] The synthesized free albumin nanoparticles were about 130 nm in size, while peptide-ICG loaded nanoparticles were around 150 nm in size and had a small polydispersity index of <0.2 (Figure 2A). The nanoparticles had a high negative charge of about -45 (Figure 2A inset). The nanoparticles were stable in PBS (pH 7.2) at RT and at 37 degrees with hydrodynamic sizes less than 200 nm up to 24 h (Figure 2B). However, when the nanoparticles were dispersed in PBS pH 5.5 (simulating the pH of lysosomes), the nanoparticles swelled up immediately showing drastic pH dependent variation in nanoparticle's stability and possible release of the payload in the endolysosomal pathway (Figure 2B). This would prevent the trapping of the neoantigen peptide in the endosomal pathway and trigger its release for effective antigen loading and presentation.

[0139] UV-Vis-NIR absorbance spectra showed that Free-ICG has an absorbance peak at 785 nm. However, when the peptide-ICG is encapsulated in albumin, the absorbance of ICG further redshifts to >800 nm (Figure 2C). This confirms the loading of peptide ICG in albumin nanoparticles. To check whether the charge of the peptide affects the size or loading of the peptide-ICG in the nanoparticles, we chose peptides that were positively, negatively or neutrally charged with similar number of lysine residues (3-4) and then used to fabricate nanoparticles. It was found that both negative and positive peptide conjugated with ICG and are loaded in albumin nanoparticles with similar loading efficiency and hydrodynamic size (Figure 2D). The synthesized nanoparticle are highly stable and remain monodisperse with sonication (Figure 2E). The nanoparticles can be prepared and stored at -20 degrees for long term, and can be redispersed in solution with sonication to form highly monodisperse nanoparticle solution.

[0140] Cytotoxicity and PDT effect of nanoparticles Before assessing the toxicity of the synthesized nanoparticles on cancer cells, free-ICG at different concentrations were incubated with MC38 and LLC1 cancer cells for 24 h before performing cell-viability assay. Free-ICG showed negligible toxicity in-vitro up to a concentration of 50 ug / ml (Figure 3A). At a concentration of 20 ug / ml, peptide-ICG loaded albumin nanoparticles also showed negligible toxicity in cancer cells (Figure 3B). While free ICG, localized mainly in the cytoplasm of mature mouse bone marrow derived dendritic cells (BMDCs), OVA- ICG loaded albumin nanoparticles localized both in the cytoplasm and outside cell membrane (Figure 3C). This shows the ability of the peptides to be successfully presented on the cell surface, when carried by the nanoparticles.

[0141] The synthesized nanoparticles were able to produce singlet oxygen following NIR irradiation in physiological solution (PBS pH 7.2) at a concentration of 30 ug / ml. Although the ability of nanoparticles to produce singlet oxygen in solution was less compared to free-ICG, the cell death followed by 6 h incubation of peptide-ICG loaded nanoparticles and NIR irradiation was significantly higher at 24 h compared to free ICG or peptide-ICG conjugate. This could perhaps be due to the significantly higher uptake of nanoparticles into the cells compared to free ICG following 6 h incubation.

[0142] To further confirm the production of reactive oxygen species (ROS) in the cell following PDT, the cells were treated with different formulation and 25 uM carboxy-H2DCFDA, a fluorogenic marker of ROS in live cells. Up on NIR irradiation there was significantly more ROS produced in cells irradiated with peptide-ICG loaded nanoparticles compared to cells with nanoparticles that were not irradiated and cells which were only irradiated with NIR (Figure 4). This confirms that the cell death is brought about by ROS induced by a combination of the photosensitizer ICG and NIR light.

[0143] Uptake kinetics and localization of nanoparticles in cancer cells

[0144] The uptake kinetics of free-ICG and peptide-ICG loaded albumin nanoparticles was studied in MC38 cancer cells. There was significantly higher ICG fluorescence in cells within 30 mins of NP incubation (Figure 5A). While free- ICG mostly localized in the cytoplasm, there was significant ICG fluorescence co-localizing the cell membrane (Figure 5B), showing the presence of the peptide on the cell-surface. This confirms that the peptide-conjugated with ICG is presented on the cell surface when delivered in albumin nanoparticle.

[0145] To further study the intracellular localization, immature immortalized mouse DCs JAWSII was incubated with 1 ug / ml Free-ICG, peptide-ICG conjugates or peptide-ICG loaded albumin nanoparticles for 3 h and co-stained with lysotracker. There was a significant uptake of nanoparticles in the DCs and the ICG fluorescence co-localized with lysotracker (Figure 6A). When the cells were co-stained with antibodies against SIINFEKL loaded H2-Kb, it was found that the staining pattern was similar to that with lysotracker (Figure 6B). This confirms that there was significantly higher loading of peptide in MHC H2-Kb in the lysosomes at 3 h. It may take slightly longer for the peptide loaded MHCs to be presented on the cell surface.

[0146] In-vivo characterization of Peptide-ICG conjugate and nanoparticles

[0147] To evaluate if the peptides would still retain ability to be loaded on the MHC and be presented to T-cells following its conjugation to ICG, the ability of free- peptides and ICG conjugated peptides to elicit antigen specific T-cells were studied. Flow cytometry results following in-vivo administration of peptides or ICG-conjugated peptides together with adjuvant CpG 1826 oligonucleotide, showed that there was no significant difference in the ability to trigger the generation of antigen specific T-cells (Figure 7A). Furthermore, ICG conjugated peptide also led significant maturation of mouse BMDCs similar to those treated with Free-peptide (Figure 7B). This confirms that the ability of peptide to be internalized by immature DCs, be loaded on the MHC, presented on the cell surface resulting in the activation of antigen specific T-cells are not hindered by the conjugation of ICG to the peptide.

[0148] The hemocompatibility of the peptide-ICG loaded albumin nanoparticles were assessed using freshly drawn mouse blood. Here, the effect of positive, negative and neutrally charged peptides in nanoparticles were also evaluated. The results revealed that the nanoparticles are hemocompatible even at higher doses (Figure 7C). Finally, a pilot study was done to assess the ability of the peptide- ICG loaded nanoparticles to trigger the generation and activation of antigen specific T-cells. Following in-vivo administration of the nanoparticles, there was significant production of antigen specific T-cells comparable to that of free- peptides (Figure 7D). This reveals that albumin nanoparticles could carry the peptide cargo effectively to the target destination and elicit strong immunogenicity.

[0149] Figure 8 illustrates detection of antigen-specific T-cell responses against tumor- associated antigens by intracellular cytokine staining (ICCS). PMA stands for Phorbol 12-myristate 13-acetate. It is a potent activator of protein kinase C that stimulates T-cells non-specifically. It is used to confirm that the T-cells are viable and capable of producing cytokines (e.g., IFN-y) when properly stimulated. In this case, PMA serves as a positive control for T-cell function. LMP2A is a latent membrane protein from Epstein-Barr virus (EBV). PMA and LMP2A are the 2 antigens used in the vaccine to showcase the immunogenicity.

[0150] MATERIALS AND METHODS

[0151] Synthesis of Peptide-ICG encapsulated Albumin nanoparticles

[0152] Conjugation of Peptides with ICG: 100 ul of 10 mg / ml peptide (dissolved DMSO) was mixed in 2 ml PBS (pH 7.2) in a glass bottle, while stirring at 1200 rpm. The pH of the solution was raised to 8.5 by adding 230 ul of IM sodium bicarbonate. Then, 60 ul of ICG-NHS-ester (10 mg / ml) was added and stirred at room temperature for 1 h. The contents of glass bottle was collected in to lo- bind centrifuge tubes and centrifuged at 16000g for 10 mins. Discard the supernatant and resuspend the pellet in 1.5ml deionised (DI) water and centrifuge at 16000g for 10 mins, Discard the supernatant.

[0153] Removal of Free-ICG: To remove unbound ICG, the pellet was resuspended in 500 ul DI water and passed through Pierce™ Dye Removal Columns (Cat no: 22858). The unbound ICG will be trapped in the resin. The loading efficiency of ICG to peptides can be then calculated from UV-Vis-NIR absorbance spectra at 785 nm (ICG absorbance peak in acetonitrile). Synthesis of Albumin nanoparticles encapsulating Peptide-ICG conjugates: The peptide-ICG conjugates are then re-dispersed in a total volume of 2ml DI water in a glass bottle. While the samples are stirring at 1000 rpm at room temperature, 400 ul of 5% recombinant human serum albumin is added dropwise. The pH of the solution is immediately raised to 9 by addition of 5 ul IN NaOH. The mixture is stirred for 30 mins, to allow the non-covalent binding of ICG into the albumin binding pockets via hydrogen and hydrophobic interactions. About 5 ml of ethanol is then added to the mixture to ethanol reduces the albumin solubility and results in self-assembly of albumin encapsulating the ICG- peptide conjugates via nanoprecipitation. 200 ul of 2.5% glutaraldehyde is then added to cross-link albumin moieties and stabilize the nanoparticles. The reaction is stirred at room temperature overnight. The nanoparticles are then collected by centrifugation (16000g for 10 mins) and water 2 times in DI water to remove excess unbound albumin, glutaraldehyde and ethanol.

[0154] Characterization of Nanoparticles

[0155] Size and charge measurement: The hydrodynamic diameter, size distribution and surface potential(zeta potential) were characterized with dynamic light scattering (DLS) at using a Zetasizer Nano-ZS90 (Malvern, UK). Size measurements were performed in different physiologically relevant solution, temperature and pH, and also to assess the stability of the nanoparticles following different storage conditions.

[0156] Concentration and Loading efficiency: The concentration of ICG and its loading in nanoparticles were obtained by measuring the absorption spectra of ICG at 785 nm or by doing an absorption scan between 600-900 nm, using a Tecan UV / Vis / NIR spectrophotometer.

[0157] Nanoparticle uptake and viability in cells: 20,000 MC38 and LLC1 mouse cancer cells were seeded in 96 well plate and allowed to attach overnight. Free-ICG or Peptide-ICG loaded albumin nanoparticles were incubated with the cells at a concentration of 20 ug / ml. For viability assay, the nanoparticles were incubated for up to 24 h and cell viability was tested by MTT assay (Promega) using manufacturer's protocol. For uptake studies in cancer cells or immature immortalized JAWSII DCs or BMDCs, 80000 -100000 cells were seeded in ibidi 8-well chambered slide. The cells were allowed to attach or acclimatize overnight. The cells were then incubated with different test formulations for 3, 6 or 24 h. The cells were then washed 1 time with PBS and incubated overnight before counterstaining with Hoechst 38222 and AlexaFluor 488 conjugated wheat germ agglutinin for staining nucleus and plasma membrane respectively. Confocal laser scanning microscopy was performed using Leica Stellaris 8 microscope using 20X or 63X oil objective lens.

[0158] Singlet oxygen testing in solution: Singlet oxygen sensor green (SOSG) was employed to monitor singlet oxygen generation following 808 nm NIR irradiation. 100 pg of SOSG was diluted in 33 pL of DMSO to achieve a final concentration of 5 mM SOSG stock. 1 pL of SOSG stock solution was added to 1 ml PBS containing 30 ug / ml ICG or Peptide-ICG albumin nanoparticles or peptide-ICG conjugate. Immediately before NIR irradiation, 100 ul of the solution was removed for baseline fluorescence measurement. The solution was then irradiated for 10 mins using a laser power of 1W, removing 100 ul samples at 5 and 10 mins. SOSG fluorescence was measured using a Tecan Sapphire plate reader using FITC excitation I emission filters.

[0159] In-vitro PDT and cell viability: 20,000 MC38 cancer cells were seeded in 96 well plate and allowed to attach overnight. Free-ICG, Peptide-ICG conjugates or Peptide-ICG loaded albumin nanoparticles were incubated with the cells at a concentration of 30 ug / ml for 6 h. The media with ICG was removed, and fresh culture media was added to the cells, before irradiating the cells with 808 nm NIR light at a power of 1 W for 2.5 mins. The cells were then allowed to rest overnight, before cell viability was tested using MTT assay (Promega).

[0160] In-vitro ROS detection: Image-iT™ LIVE Green Reactive Oxygen Species Detection Kit (Cat#I36007, Molecular Probes) was used to detect the production of ROS in MC38 cells. Briefly, MC38 cells in 8-well chambered slides were incubated with various formulations for about 6 h. The cells were then washed and treated with 25 uM carboxy-H2DCFDA for 30 mins at 37 degrees. The cells were then irradiated to induce ROS. The positive control was treated as per manufacturer's instruction. Live imaging was done using Olympus FV3000 Scanning Confocal Microscope.

[0161] Immunofluorescence staining: 80,000 JAWSII immortalized immature mouse DCs were seeded in ibidi 8-well chambered slide and allowed to attach overnight. Free-ICG or Peptide-ICG loaded albumin nanoparticles were incubated with the cells at a concentration of 1 ug / ml for 3 h. The cells were then washed twice with IX PBS and fixed using 10% neutral buffered formalin (NBF) for 20 mins. Following washing with IX PBS, the cells were blocked with 0.2% BSA in PBS-Tween, 1 h at room temperature. The cells were then stained using PE conjugated anti-mouse H-2Kb bound to SIINFEKL Antibody(Clone:25- D1.16, Biolegend) in blocking buffer at a dilution of 1 :200, overnight at 4 degrees. The cells were then washed with PBS and counterstained with Hoechst 38222 and AlexaFluor 488 conjugated wheat germ agglutinin for staining nucleus and plasma membrane respectively. Confocal laser scanning microscopy was performed using Leica Stellaris 8 microscope using 20X or 63X oil objective lens, using filter set for AF790 and Rhodamine for ICG and PE respectively.

[0162] Lysosome staining : 80000 JAWSII cells grown on ibidi 8-well chambered slides were treated with different peptide formulations for 3 h at a concentration of 1 ug / ml at 37 degrees. In the last 1 h of incubation, 200 nM Lysotracker red DND99 (Invitrogen™). The cells were washed with IX PBS 3times, and fixed with 10% neutral buffered formalin (NBF) for 20 mins. The cells were then washed with PBS and counterstained with Hoechst 38222 and AlexaFluor 488 conjugated wheat germ agglutinin for staining nucleus and plasma membrane respectively. Confocal laser scanning microscopy was performed using Leica Stellaris 8 microscope using 20X or 63X oil objective lens, using filter set forAF790 and TRITC for ICG and Lysotracker respectively.

[0163] Hemolysis assay: Female balb / c nude mice, 15 weeks of age, weighing an average of 17 g were obtained from Invivos Singapore. Fresh blood (~1 mL) was obtained from mice via cardiac puncture. All procedures carried out in this study are approved by the Institutional Animal Care and Use Committee (IACUC), A*STAR, Singapore and were conducted in accordance with international standards. Red blood cells (RBCs) were separated from plasma by centrifuging at 1500 rpm for 15 min at 4°C. The isolated RBCs were further washed three times with sterile PBS by centrifugation until the supernatant was clear, and resuspended in 2 mL of PBS. Then 100 pL of peptide-ICG loaded NP suspension in PBS at concentrations ranging from 150-300 ug / ml ICG (300-600 ug / ml peptide) were added to 100 pl of the RBCs suspension. Following a 2 h at 37°C under occasional shaking, the suspensions were centrifuged at 1500 rpm for 15 min. Subsequently, 100 pL of supernatant from each centrifuge tube was used to analyze hemoglobin release by microplate reader at the wavelength of 576 nm. Control experiments were performed under the same experimental conditions, where 100 pL of the RBCs suspension was added to 100 pL of PBS as a negative control and to 100 pL of 0.2% Triton X-100 as a positive control. The percentage hemolysis was calculated using the following equation: Hemolysis (%) = (OD576 sample - OD576 negative control) / (OD576 positive control - OD576 negative control) X100%.

[0164] BMDC culture and maturation: Immature BMDCs were collected from the femurs of C57BL / 6 female mice (15 weeks old). After 7 days of culture in complete RPMI media supplemented with 20 ng / ml mouse GM-CSF, BMDCs were seeded in 24-well plates (1 x 106 cells per well) and treated with free-peptide, peptide- ICG conjugates, free-ICG or CpG alone for 24 h. BMDCs were labelled with anti- CDllc, anti-CD86, anti-MHC II, and anti-CD80. The expression level of surface markers was detected by BD LSR II flow cytometer.

[0165] In-vivo antigen specific T-cells induction: 7-8 weeks old C57BL / 6 female mice were immunized by subcutaneous hock injection with various formulations with 200 ug peptide and 30 ug CpG 1826 ODN per injection per mice. The immunization was repeated 1 week later. On day 14, spleens were harvested and 3 X106 splenocytes were labelled with PE conjugated H-2 Kb -SIINFEKL Dextramer (Immudex), anti- mouse CD3, CD4 and CD8 antibodies (Miltenyi Biotech), following manufacturer's instruction and expression of the markers was detected by BD LSR II flow cytometer. It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

[0166] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0167] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0168] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

Claims1. An albumin nanoparticle, comprising : a) a neoantigen peptide conjugated to an albumin hitchhiking compound to form a conjugated compound; and b) a plurality of albumins; wherein the conjugated compound is non-covalently bonded to the plurality of albumin via the albumin hitchhiking compound; wherein the plurality of albumins are non-covalently bonded and covalently crosslinked to each other; and wherein the conjugated compound is configured to be released from the albumin nanoparticle when subjected to pH 6 and below.

2. The albumin nanoparticle according to claim 1, wherein the neoantigen peptide is characterised by an amino acid chain length of 20 residues to 40 residues.

3. The albumin nanoparticle according to claim 1 or 2, wherein the neoantigen peptide is characterised by an amino acid chain length of 25 residues to 30 residues.

4. The albumin nanoparticle according to any one of claims 1 to 3, wherein the neoantigen peptide comprises a shared hot spot mutation.

5. The albumin nanoparticle according to any one of claims 1 to 4, wherein the albumin hitchhiking compound is a fluorophore configured to be excitable under near-infrared (NIR) irradiation.

6. The albumin nanoparticle according to any one of claims 1 to 5, wherein the albumin hitchhiking compound is selected from indocyanine green (ICG).

7. The albumin nanoparticle according to any one of claims 1 to 6, wherein the neoantigen peptide is conjugated to an albumin hitchhiking compound via an amide bond.

8. The albumin nanoparticle according to any one of claims 1 to 7, wherein the conjugated compound is characterised by a mole ratio of neoantigen peptide to albumin hitchhiking compound of about 1 : 1 to about 5: 1.

9. The albumin nanoparticle according to any one of claims 1 to 8, wherein the albumin nanoparticle is characterised by a percent loading of neoantigen peptide of about 2 %wt / wt to about 30 %wt / wt.

10. The albumin nanoparticle according to any one of claims 1 to 9, wherein the albumin nanoparticle is characterised by a percent loading of albumin hitchhiking compound of about 2 %wt / wt to about 30 %wt / wt.

11. The albumin nanoparticle according to any one of claims 1 to 10, wherein the conjugated compound is non-covalently bonded to the plurality of albumins via electrostatic interaction, van Der Waals forces, and / or hydrogen bonding.

12. The albumin nanoparticle according to any one of claims 1 to 11, wherein the neoantigen peptide is configured to be cleaved from the albumin hitchhiking compound when subjected to pH 6 and below.

13. The albumin nanoparticle according to any one of claims 1 to 12, wherein the plurality of albumins are non-covalently bonded to each other via hydrophobic interaction, hydrogen bonding, and / or a reduced solubility of albumin induced by a non-solvent.

14. The albumin nanoparticle according to any one of claims 1 to 13, wherein the crosslink is configured to cleave when subjected to pH 6 and below.

15. The albumin nanoparticle according to any one of claims 1 to 14, wherein the plurality of albumins are covalently crosslinked to each other via a glutaraldehyde linker.

16. The albumin nanoparticle according to any one of claims 1 to 15, whereinthe plurality of albumins are covalently crosslinked to each other via their lysine residues thereof.

17. The albumin nanoparticle according to any one of claims 1 to 16, wherein the albumin nanoparticle is characterised by a percent crosslink of about 50% to about 80%.

18. The albumin nanoparticle according to any one of claims 1 to 17, wherein the albumin nanoparticle is characterised by a particle size of about 90 nm to about 400 nm.

19. The albumin nanoparticle according to any one of claims 1 to 18, wherein the albumin nanoparticle is characterised by a particle size of about 150 nm.

20. The albumin nanoparticle according to any one of claims 1 to 19, wherein the albumin nanoparticle is characterised by a polydispersity index of less than about 0.4.

21. The albumin nanoparticle according to any one of claims 1 to 20, wherein the albumin nanoparticle is characterised by a zeta potential of about -10 mV to about -80 mV.

22. The albumin nanoparticle according to any one of claims 1 to 21, wherein the albumin nanoparticle is characterised by a zeta potential of about -45 mV.

23. A pharmaceutical composition comprising the albumin nanoparticle according to any one of 1 to 22, and optionally an excipient.

24. A method of synthesizing an albumin nanoparticle, comprising: a) conjugating a neoantigen peptide with an albumin hitchhiking compound to form a conjugated compound; b) mixing the conjugated compound with albumin in order for the conjugated compound to non-covalently bind to albumin; and c) nanoprecipitating and crosslinking the albumin of step b) in order to formthe albumin nanoparticles.

25. A method of delivering a neoantigen peptide to a subject in need thereof, comprising administering a therapeutic amount of the albumin nanoparticle according to any one of claims 1 to 22 to the subject.

26. A method of treating and / or preventing a cancer in a subject in need thereof, comprising administering a therapeutic amount of the albumin nanoparticle according to any one of claims 1 to 22 to the subject.

27. The method according to claims 25 or 26, wherein the cancer is selected from nasopharyngeal carcinoma, lung cancer, breast cancer, colorectal cancer, melanoma, and a combination thereof.

28. The method according to any one of claims 25 to 27, wherein the method further comprises a step of photothermally treating the cancer using near-infra red (NIR) irradiation.

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